P-glycoprotein (P-gp) belongs to a superfamily of ABC transporters found both in pro- and eucaryotes that act as energy-dependent efflux pumps for transporting a wide variety of low molecular weight compounds out of cells (for a review see reference 1 of Appendix A). ABC transporters are a constantly growing superfamily of membrane proteins found in bacteria through humans. ABC transporters use energy of ATP hydrolysis for transport of a wide spectrum of compounds (e.g., anticancer drugs, toxins, antibiotics, and lipids) from cells. In mammals, this superfamily includes, in addition to the P-gp transporters (MDR1 and MDR3 in humans), the MRP subfamily (already including 6 members), and several other proteins (e.g., LRP and BCRP) (2,3).
The majority of ABC transporters have an ability to recognize and efflux numerous substrates of divergent chemical structure, including many anticancer drugs, from cells and tissue. The molecular mechanisms underlying broad substrate specificity of ABC transporters are generally unknown (3). The expression of ABC transporters results in cross resistance of cells to numerous toxic compounds, known as multidrug resistance (MDR) (1,3,4).
Increased expression of ABC transporters in tumor cells is one of the major mechanisms of cancer resistance to chemotherapy (1-3). Although the clinical relevance of multidrug resistance is debated, P-gp and other ABC transporters are viewed as targets for therapeutic suppression to increase the susceptibility of multidrug resistant cancers to chemotherapy (5).
The understanding of the normal physiological role of ABC transporters is derived from an analysis of phenotypes of mice genetically deficient in the genes encoding these proteins. Mice lacking both mdr1a and mdr1b genes (i.e., two homologs of human P-gp-encoding MDR1 gene with different tissue expression (6)) develop normally, but were found to be extremely sensitive to particular xenobiotics and have strong alterations in pharmacokinetics of drugs known to be P-gp substrates (7,8). Moreover, a deficiency in P-gp was associated with the ability of P-gp substrates to pass through blood-brain barrier (6,7,8).
In mammals, and particularly in humans, the expression of ABC transporters is restricted to specific organs, including the intestine, kidney, liver, endothelia of brain, testis, and placenta, which is consistent with role of these organs in general detoxification and in establishment of blood-brain, blood-testis, and placental barriers (6). Although no physiological abnormalities were observed in the mice deficient in mpr1 gene, the mice did demonstrate increased sensitivity to particular toxins and changes in glutathione metabolism (9). Robbiani et al. also reported possible involvement of MRP1 in dendritic cells migration to lymph nodes (10).
Some members of the superfamily of ABC transporters apparently have a more narrow and specific spectrum of substrates, e.g., targeted disruption of mdr2 gene in mice results in a deviation in phosphatidylcholine and other phospholipid excretion in a bile (11). Thus, in addition to an ABC supertransporter role in cancer resistance to chemotherapy, ABC transporters are involved in numerous physiological processes and their value as therapeutic targets is envisioned as far broader than cancer treatment.
Over the last several years, chemical and protein inhibitors of ABC transporters have been developed to overcome MDR, and many of the inhibitors have been tested in clinical trials. However, there is a need not only for inhibitors of ABC transporters, but also for inducers of their activity and modulators of their substrate specificity. This need stems from the natural function of these proteins, which is known to be involved in protection of cells and tissues of an organism from cytotoxic compounds. Thus, compounds having such modulating properties can be used to facilitate detoxification of cells and tissues under conditions of acute or chronic poisoning.
It was previously found (5) that mutations of P-gp, the best studied ABC transporter, can strongly alter substrate specificity, thereby changing the pattern of cross resistance of cells expressing mutant P-gp, presumably by changing protein conformation. The present invention is directed to a similar effect caused by compounds capable of modulating multidrug transporting activity or modulating substrate specificity of multidrug transporters.